Mycobacterium avium subspecies paratuberculosis vaccines and methods of using the same

ABSTRACT

The invention relates to vaccine compositions composed of at least one  Mycobacterium avium  subspecies  paratuberculosis  (MAP) antigen, or attenuated or killed MAP for use in methods of immunizing a human against a MAP infection, preventing or treating a MAP infection, and preventing a human disease associated with a MAP infection.

INTRODUCTION

This application claims benefit of priority to U.S. Provisional Patent Application Ser. No. 60/913,315, filed Apr. 23, 2007, the content of which is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

Johne's disease is a chronic diarrheal enteric disease in ruminants that is caused by Mycobacterium avium subspecies paratuberculosis (MAP)(Johne & Frothingham (1895) Dtsch. Zeitschr. Tiermed. Vergl. Pathol. 21:438-454). Live MAP is shed into the milk of cows with Johne's disease (Sweeney (1996) Vet. Clin. North Am. Food Anim. Pract. 12(2):305-12). MAP has been cultured from commercially available pasteurized milk in Europe and the United States (Grant (1998) Appl. Environ. Microbiol. 64(7):2760-1; Ellingson, et al. (2005) J. Food Prot. 68(5):966-72). When Crohn's disease was first described (Crohn, et al. (1932) J. Amer. Med. Assoc. 99:1323-1328), similarities to Johne's disease were identified (Dalziel (1913) Br. Med. J. ii:1068-1070). However, in humans MAP exists in the cell wall-deficient form (Chiodini (1987) J. Clin. Microbiol. 25:796-801). Therefore, in the early analysis of Crohn's disease, MAP could not be detected in humans by the mycobacterial identification techniques of the time, because such techniques stained the mycobacterial cell wall (Ziehl (1882) Dtsch. Med. Wschr. 8:451; Neelsen (1883) Zbl. Med. Wiss. 21:497-501). However, since 1913 the presence of MAP has been identified in humans by other means (see, e.g., Greenstein (2003) Lancet Infect. Dis. 3(8):507-14) and an infectious etiology has been posited for some (Hermon-Taylor (1998) Ital. J. Gastroenterol. Hepatol. 30(6):607-10; Borody, et al. (2002) Dig. Liver Dis. 34(1):29-38), or all (Greenstein (2005) Genetics, Barrier Function, Immunologic & Microbial Pathways. Munster, Germany:25) of inflammatory bowel disease (IBD).

Since the first detection of MAP RNA (Mishina, et al. (1996) Proc. Natl. Acad. Sci. USA 93(18):9816-9820), MAP has been suggested as being the primary and unique, etiological agent of all IBD (Naser, et al. (2004) Lancet 364(9439):1039-1044; Autschbach, et al. (2005) Gut 54(7):944-9; Greenstein (2005) supra; Greenstein (2005) Genetics, Barrier Function, Immunologic & Microbial Pathways. Munster, Germany:24; Greenstein (2005) Crohn's and Colitis Foundation (CCFA) National Research and Clinical Conference. Fourth Annual Advances in Inflammatory Bowel Disease. Miami, Fla.:211) including Perforating and Non-perforating Crohn's disease (Greenstein, et al. (1988) GUT 29:588-592; Gilberts, et al. (1994) Proc. Natl. Acad. Sci. USA 91(126):12721-12724) and ulcerative colitis. It is believed that the particular clinical presentation of IBD that manifests is dependent upon the infected individual's immune response to MAP (Gilberts, et al. (1994) supra) This is analogous to another mycobacterial disease, leprosy. There are two clinical forms of leprosy, tuberculoid and lepromatous (Hansen (1874) Norsk Magazin Laegevidenskaben 4:1-88), both of which are caused by the same organism, M. leprae. The form of leprosy that manifests in a given individual is determined by the immune response of the infected patient (Yamamura, et al. (1991) Science 254:277-279), not by the phenotype or genotype of the leprosy bacillus.

It has been suggested that Koch's postulates (Koch (1882) Berl. Klin. Wschr. 19:221-230), originally promulgated for use in demonstrating tuberculosis infection, may have been met for MAP in Crohn's disease (Greenstein (2003) supra) and more recently for MAP in ulcerative colitis (Greenstein (2005) supra; Naser, et al. (2004) supra).

The link between MAP infection and other diseases is under investigation. An association between ulcerative colitis and Multiple Sclerosis has been suggested (Rang, et al. (1982) The Lancet pg. 555) and the positive association between IBD incidence rates and Multiple Sclerosis has led to the suggestion that these two chronic, immunologically-mediated diseases may have a common environmental etiology (Green, et al. (2006) Am. J. Epidemiol. 164(7):615-23). However, the common causal agent of ulcerative colitis and Multiple Sclerosis was not identified. Moreover, while the symptoms of Multiple Sclerosis have been ameliorated with variety of therapeutic agents including azathioprine, methotrexate, cyclophosphamide and mitoxantrone (Kaffaroni, et al. (2006) Neurol. Sci. 27 Suppl. 1:S13-7), which have been suggested to mediate the secondary inflammatory response, there has been no indication that these agents affect the primary etiological agent.

There is increasingly compelling evidence that MAP may be zoonotic (Greenstein & Collins (2004) Lancet 364(9432):396-7) and a human pathogen in gastrointestinal disease (Greenstein (2005) supra) and other diseases as well. There is an additional indication that in man, MAP is systemic and not confined to the gastrointestinal tract (Naser, et al. (2000) Am. J. Gastroenterol. 95(4):1094-5; Naser, et al. (2004) Lancet 364(9439):1039-1044). It is suggested that the reason MAP is zoonotic and has been missed as an etiological agent is that the medical profession has been unknowingly treating MAP with anti-inflammatory agents (e.g., 5-amino salicylic acid, methotrexate, and 6-mercaptopurine), which in fact have anti-MAP activity (Greenstein, et al. (2007) PLoS ONE 2:e161; Greenstein, et al. American Society of Microbiology 2007, Toronto, Canada).

Given this identified association between MAP as the etiological agent for a variety of human diseases, there is a need in the art for compositions and methods for preventing MAP infection. The present invention meets this need by providing vaccines for the prevention of a MAP infection and MAP-associated diseases.

SUMMARY OF THE INVENTION

The present invention is a method for producing a vaccine for immunizing a human against a Mycobacterium avium subspecies paratuberculosis (MAP) infection. The method involves admixing at least one MAP antigen, or attenuated or killed MAP with a suitable carrier thereby producing a vaccine for the immunization of a human against a MAP infection. In one embodiment, the attenuated or killed MAP is cell wall-competent or cell wall-deficient. In another embodiment, the MAP antigen is GroES, AhpD, 32 kDa antigen, 34 kDa antigen, 34.5 kDa antigen, 35 kDa antigen, 36 kDa antigen, 42 kDa antigen, 44.3 kDa antigen, AhpC antigen or 65 kDa antigen.

Methods for immunizing a human against a MAP infection, preventing or treating a MAP infection, and preventing a human disease associated with a MAP infection are also provided.

DETAILED DESCRIPTION OF THE INVENTION

Epidemiological analysis identifies a parallelism in the increasing incidence of Crohn's disease, ulcerative colitis and Multiple Sclerosis (Green, et al. (2006) supra). In Alzheimer's disease the use of “anti-inflammatories” shows therapeutic benefit (Rogers, et al. (1993) Neurology 43(8):1609-11). Additionally, there is the suggestion that rheumatoid arthritis is protective against Alzheimer's disease (McGeer, et al. (1990) Lancet 335(8696):1037). Analogous to lepromatous leprosy (Hansen (1874) Norsk Magazin for Laegevidenskaben 4:1-88) and tuberculoid leprosy, it is now posited that Multiple Sclerosis and perforating Crohn's disease (Gilberts, et al. (1994) Proc. Natl. Acad. Sci. USA 91(126):12721-12724) are the “acute” forms of a Mycobacterium avium subspecies paratuberculosis (MAP; basonym M. paratuberculosis) infection, whereas Alzheimer's Disease and obstructive Crohn's or ulcerative colitis are the chronic forms of a MAP infection. It is further posited that a causative relationship between MAP and diseases such as IBD and Multiple Sclerosis have been missed because it has not been appreciated that standard “immunomodulatory” treatment regimes, whose mechanisms of actions are unknown or speculated upon, are in fact effective because they are treating a MAP infection. It is posited that MAP is also responsible for a variety of diseases where an infectious etiology has been suggested, e.g., sarcoidosis, ankylosing spondylitis, psoriasis, and psoriatic arthritis and rheumatoid arthritis. Coincidentally, these diseases are often treated with “immunomodulatory” and “anti-inflammatory” agents that have now been shown to interfere with the growth kinetics of MAP.

While some reports have indicated that high-temperature short-time pasteurization does not effectively kill MAP in milk (Grant, et al. (1998) Lett. Appl. Microbiol. 26:166-170; Grant, et al. (1999) Lett. Appl. Microbiol. 28:461-465), killing by turbulent-flow conditions has been demonstrated (Stabel, et al. (1997) Appl. Environ. Microbiol. 63:4975-4977). Given the identification of potential sources of infection and that MAP is widespread over the industrialized as well as non-industrialized world and, a multipronged approach including vaccines, antibiotics, and public health measures are needed to control and prevent MAP infections. Accordingly, having appreciated that MAP may be the etiological agent of a plurality of human diseases or conditions, the present invention provides vaccines and methods for immunizing human subjects against a MAP infection.

For the purposes of the present invention, a vaccine of is intended to include whole MAP cells, either cell wall-competent or cell wall-deficient; MAP cell extracts; isolated protein (i.e., a subunit vaccine); or combinations thereof.

Whole cell vaccines can be produced from cell well-competent and/or cell wall-deficient MAP which has been inactivated or attenuated or has been killed. Live attenuated vaccines have the advantage of mimicking the natural infection enough to trigger an immune response similar to the response to the wild-type organism. Such vaccines generally provide a high level of protection, especially if administered by a natural route, and some may only require one dose to confer immunity. Because MAP exists in humans in the cell wall-deficient state, a vaccine which targets this obligate intracellular form is desirable. By way of illustration, cell wall-competent and cell wall-deficient (i.e., spheroplasts) vaccine preparations have been shown to reduce lesion scores associated with Johne's Disease in baby goats (Hines, et al. (2005) 8^(th) International Colloquium on Paratuberculosis, Copenhagen, Denmark). MAP can be attenuated using any conventional strategy employed in producing an attenuated M. tuberculosis. For example, serial passage or culture of the active organism in culture media or cells can be employed to attenuate MAP. Alternatively, the vaccine of the present invention can contain heat-killed MAP cells. In this regard, vaccination of calves with a heat-killed field strain of MAP results in high concentrations of IFN-γ and better protection against a MAP challenge exposure than does a commercially available vaccine (Uzonna, et al. (2002) Proc. 7^(th) Intl. Coll. Paratuberculosis; Juste (ed)).

In addition, or as an alternative to an attenuated or killed MAP vaccine, a MAP subunit vaccine can be employed. Any one of the well-known MAP-specific antigens, or antigen fragments thereof, commonly employed in veterinary medicine can be used as a vaccine in accordance with the present invention. See Table 1.

TABLE 1 SEQ Size ID MAP protein Characteristic (kDa) NO: GroES Heat shock protein 10 1 AhpD Alkyl hydroperoxide reductase D 19 2 32-kDa antigen Fibronectin binding properties, 32 secreted 34-kDa antigen Cell wall antigen, B-cell epitope 34 3 34-kDa antigen Serine protease 34 4 34.5-kDa antigen Cytoplasmic protein 34.5 35-kDa antigen Immunodominant protein 35 36-kDa antigen p36 antigen 36 5 42-kDa antigen Cytoplasmic protein 42 44.3-kDa antigen Soluble protein 44.3 AhpC Alkyl hydroperoxide reductase C 45 6 65-kDa antigen GroEL heat shock protein 65 7

The 32-kDa secreted protein with fibronectin binding properties has been implicated in protective immunity (Andersen, et al. (1991) Infect. Immun. 59:1905-1910; El-Zaatari, et al. (1994) Curr. Microbiol. 29:177-184) and the 34-kDa cell wall antigenic protein is homologous to a similar immunogenic protein in M. leprae (De Kesel, et al. (1992) Scand. J. Immunol. 36:201-212; De Kesel, et al. (1993) J. Clin. Microbiol. 31:947-954; Gilot, et al. (1993) J. Bacteriol. 175:4930-4935; Silbaq, et al. (1998) Infect. Immun. 66:5576-5579). The seroreactive 34-kDa serine protease expressed in vivo by MAP has also been described (Cameron, et al. (1994) Microbiology 140:1977-1982; however, this antigen is different from the 34-kDa antigen described above. Another strongly immunoreactive protein of 35 kDa has also been identified in M. avium complex isolates, including MAP (El-Zaatari, et al. (1997) J. Clin. Microbiol. 35:1794-1799). A more thoroughly characterized protein of 65 kDa from MAP is a member of the GroEL family of heat shock proteins (El-Zaatari, et al. (1994) Curr. Microbiol. 29:177-184; El-Zaatari, et al. (1995) Clin. Diagn. Lab. Immunol. 2:657-664). Like the GroES proteins, the GroEL antigens from other mycobacteria are highly immunogenic (Shinnick (1987) J. Bacteriol. 169:1080-1088; Thole, et al. (1987) Infect. Immun. 55:1466-1475; Thole, et al. (1988) Infect. Immun. 56:1633-1640).

The alkyl hydroperoxide reductases C and D (AhpC and AhpD) have also been characterized as immunogenic proteins of MAP (Olsen, et al. (2000) Infect. Immun. 68:801-808). Unlike other mycobacteria, large amounts of these antigens are produced by MAP when the bacilli are grown without exposure to oxidative stress. AhpC is the larger of the two proteins and appears to exist as a homodimer in its native form since it migrates at both 45 and 24 kDa under denaturing conditions. In contrast, AhpD is a smaller monomer, with a molecular mass of about 19 kDa. Antiserum from rabbits immunized against AhpC and AhpD reacted only with MAP proteins and not with proteins from other mycobacterial species, indicating that antibodies against these proteins are not cross-reactive. Furthermore, peripheral blood monocytes from goats experimentally infected with MAP were capable of inducing gamma interferon (IFN-γ) responses after stimulation with AhpC and AhpD, confirming their immunogenicity (Olsen, et al. (2000) Infect. Immun. 68:801-808).

Antigenic proteins disclosed herein, can be prepared and purified by any conventional method including recombinant production. The term purified does not require absolute purity; rather, it is intended as a relative definition, and can include preparations that are highly purified or preparations that are only partially purified.

Vaccines of the present invention are prepared using routine methods. Generally, vaccines are prepared as injectables, in the form of aqueous solutions or suspensions. Vaccines in an oil base are also well-known such as for inhaling. Solid forms which are dissolved or suspended prior to use can also be formulated. Suitable carriers, diluents and excipients are generally added that are compatible with the active ingredients and acceptable for use in humans. Examples of such carriers include, but are not limited to, water, saline solutions, dextrose, or glycerol. Carriers can also include liposomes or microspheres. Combinations of carriers can also be used. A generally recognized compendium of methods and ingredients of vaccine compositions is Remington: The Science and Practice of Pharmacy, Alfonso R. Gennaro, editor, 20th ed. Lippincott Williams & Wilkins: Philadelphia, Pa., 2000.

Vaccine compositions can further incorporate additional substances to stabilize pH, or to function as adjuvants, wetting agents, or emulsifying agents, which can serve to improve the effectiveness of the vaccine. Examples of suitable adjuvants include, but are not limited to, aluminum salts; Incomplete Freund's adjuvant; threonyl and n-butyl derivatives of muramyl dipeptide; lipophilic derivatives of muramyl tripeptide; monophosphoryl lipid A; 3′-de-O-acetylated monophosphoryl lipid A; cholera toxin; QS21; phosphorothionated oligodeoxynucleotides with CpG motifs and adjuvants disclosed in U.S. Pat. No. 6,558,670.

Vaccines are generally formulated for parenteral administration and are injected either subcutaneously or intramuscularly. Vaccines can also be formulated as suppositories or for oral or nasal administration using methods known in the art.

The amount of vaccine sufficient to confer immunity to pathogenic MAP is determined by methods well-known to those skilled in the art. This quantity will be determined based upon the characteristics of the vaccine recipient and the level of immunity required. Typically, the amount of vaccine to be administered will be determined based upon the judgment of a skilled physician. Where vaccines are administered by subcutaneous or intramuscular injection, a range of 0.5 to 500 μg purified protein can be given.

The present invention is also directed to a vaccine in which an antigenic protein, or antigenic fragment thereof, is delivered or administered in the form of a polynucleotide encoding the protein or fragment (i.e., a DNA vaccine). In DNA vaccination, the patient is administered a polynucleotide encoding a antigenic protein that is then transcribed, translated and expressed in some form to produce strong, long-lived humoral and cell-mediated immune responses to the antigen. The polynucleotide can be administered using viral vectors or other vectors, such as liposomes, and can be combined with a acceptable carrier.

In addition, the proteins of the present invention can be used as antigens to stimulate the production of antibodies for use in passive immunotherapy, for use as diagnostic reagents, and for use as reagents in other processes such as affinity chromatography.

The present invention also embraces a method of using the presently disclosed anti-MAP vaccine as a means of immunizing animals, especially mammals, most particularly humans, against a MAP infection. In accordance with such a method, a MAP vaccine containing at least one MAP antigen, or attenuated or killed MAP is administered to a human subject in an amount effective to stimulate a measurable immune response. A measurable immune response can include a humoral response (e.g., production of antibodies to a particular antigen) or cell-mediate immune response (e.g., elicitation of a T cell response as determined by the production of cytokines such as IFN-gamma or IL-10).

In so far as the vaccine disclosed herein can be used to immunize a human against a MAP infection, the present invention also provides for a method of preventing or treating a MAP infection, as well as a human disease associated with a MAP infection. Such methods involve administering to a human subject, a vaccine containing at least one MAP antigen, or attenuated or killed MAP, as disclosed herein, in an amount effective to prevent or attenuate said MAP infection or symptoms of the MAP-associated disease. In using the methods of the invention, the disease to be prevented or treated is desirably ulcerative colitis, irritable bowel syndrome, Crohn's Disease, Multiple Sclerosis, Alzheimer's Disease, sarcoidosis, ankylosing spondylitis, psoriasis, psoriatic arthritis or rheumatoid arthritis.

The invention is described in greater detail by the following non-limiting examples.

EXAMPLE 1 Anti-MAP Vaccine

Mice (10 per group, e.g., wild-type or IL-18 deficient mice (Momotani, et al. (2002) Proc. 7^(th) Intl. Coll. Paratuberculosis, Juste (ed)) are immunized intraperitoneally (i.p.) with either AhpC or AhpD protein (15 μg in 50 μl PBS (phosphate-buffered saline) emulsified in 50 μl complete Freund's adjuvant (CFA)). A group of 10 mice are sham-immunized with PBS and CFA only.

A second immunization of 15 μg protein with incomplete Freund's adjuvant (IFA) is administered 3 weeks later (with the sham-immunized group receiving PBS and IFA).

Blood is drawn at weeks 5 and 7. Sera from each group are pooled for analysis of anti-AhpC and anti-AhpD antibody by ELISA. Mice are challenged at week 8 by intraperitoneal injection of MAP. Mice are monitored for signs and symptoms of disease.

Data will indicate that immunization of mice with either recombinant AhpC or AhpD proteins elicits a response capable of protecting against MAP infection.

EXAMPLE 2 Immunogenicity of Anti-MAP Vaccine in Humans

Sera from patients with culture-proven MAP infection are used in western blot analysis containing recombinant AhpC or AhpD protein.

The results of this analysis will demonstrate that sera from patients with MAP infections exhibit reactivity with either AhpC or AhpD, thereby indicating that AhpC and AhpD are recognized by the human immune system and suggest that antibodies able to bind the AhpC or AhpD protein can be produced during natural MAP infection in humans. Further, this data provides evidence that AhpC and AhpD are expressed in vivo by MAP during infection, and thus can be available as targets for immunoprophylaxis, immunotherapy, or to provide immune responses in subjects vaccinated with these proteins. 

1. A method for producing a vaccine for immunizing a human against a Mycobacterium avium subspecies paratuberculosis (MAP) infection comprising admixing at least one MAP antigen, or attenuated or killed MAP with a suitable carrier thereby producing a vaccine for the immunization of a human against a MAP infection.
 2. The method of claim 1, wherein the attenuated or killed MAP is cell wall-competent or cell wall-deficient.
 3. The method of claim 1, wherein the MAP antigen is GroES, AhpD, 32 kDa antigen, 34 kDa antigen, 34.5 kDa antigen, 35 kDa antigen, 36 kDa antigen, 42 kDa antigen, 44.3 kDa antigen, AhpC antigen or 65 kDa antigen.
 4. A method for immunizing a human against a MAP infection comprising administering to a human subject a vaccine containing at least one MAP antigen, or attenuated or killed MAP thereby immunizing the human against a MAP infection.
 5. A method for preventing or treating a MAP infection comprising administering to a human subject a vaccine containing at least one MAP antigen, or attenuated or killed MAP thereby preventing or treating a MAP infection.
 6. A method for preventing a human disease associated with a MAP infection comprising administering to a human subject a vaccine containing at least one MAP antigen, or attenuated or killed MAP thereby preventing a human disease associated with a MAP infection. 